The pathway of steroid hormone biosynthesis in the adrenal cortex is composed of 5 to 6 different enzymatic steps. The intracellular distribution of the enzymes composing this pathway is unusual and of interest: about half are associated with the mitochondria and half with the endoplasmic reticulum. The reasons for this distribution are unknown but two important consequences can be stated: (1) each enzyme is subjected to and regulated by a milieu unique to the associated organelle, and (2) mechanisms must exist for the transport and retention of the steroid hormone precursor within the adrenal cortex cell. The long-rang objective of this proposed research will be to define these controls and mechanisms. The research will focus on 3 beta-hydroxysteroid dehydrogenase/isomerase (HSD), an enzyme which we have shown to have a dual mitochondrial and microsomal localization and with a pregnenolone-binding protein that we have described in the cytosol, mitochondria and microsomes. HSD is an important branch point enzyme of adrenal steroidogenesis. Control of the activity of HSD is crucial for the proper channeling of pregnenolone toward either the glucocorticoids or dehydroepiandrosterone and the adrenal androgens. The immediate aims are (1) to learn how the kinetic properties of HSD are regulated by the organelle with which it is associated, and (2) to determine the role of the pregnenolone-binding protein in the transport and retention of pregnenolone in adrenal cortex cells. Subcellular fractions, proteins and enzymes isolated from rat calf and human adrenal cortices will be studied using the techniques of enzymology and organelle, suborganelle and protein biochemistry.